Development of technology for culturing target cells or tissue, while maintaining the normal form and functions thereof, is indispensable for advancing the so-called regenerative medicine. Thus, development of tissue engineering that reproduces the target tissue by systematic culturing of cells is especially anticipated.
One of the tasks in the field of tissue engineering is to establish a method for spatially culturing (three-dimensionally culturing) cells (in particular ES cells or other stem cells) or cell masses (including partially ordered aggregates; same hereinbelow) in a state in which a function of differentiating (aggregating) into a target tissue or organ is maintained, and also a culture vessel that is suitable for such method. With a general two-dimensional culturing method, for example, a method for culturing cells or tissue on a planar Petri dish, the cultured material is affected by gravity and easily assumes the form of a thin sheet, and such method is unsuitable for culturing a tissue that is wished to function properly in an organism that is a three-dimensional environment.
Further, establishing a method for culturing stem cells and cell masses (three-dimensional culturing method) and a culture vessel therefore are also important in terms of clarifying the mechanism of differentiation (aggregation) of ES cells (embryonic stem cells) and other stem cells. In particular, because most of the animal cells are adherent cells, long-term culturing in a floating state is difficult to perform by the conventional methods.
Conventionally, three-dimensional culturing of cells of this kind has been conducted by using a variety of culture carriers such as glass beads, metal chips, and inorganic porous materials (Japanese Patent Application Laid-open No. 2004-267562). However, these carriers are limited to those that do not inhibit multiplication of target cells (tissue). In addition, the configuration of culture device is made complex. Further, in three-dimensional culturing using a carrier, the carrier and culture are difficult to separate and culturing large quantities of cells and tissue having identical form and functions is generally difficult.
On the other hand, a hanging drop method (HD method) is a conventional method that uses no carrier. With the HD method, a culture medium has a semispherical shape, thereby making it possible to cause contact and aggregation of cells in the culture medium and form a three-dimensional cell mass. However, semispherical culture medium that can be formed by the HD method are limited to a small size. Further, the HD method requires cautious operations, and the culturing success ratio is not more than about 60% even for a skilled operator.
A method using a vessel with a U-shaped bottom (typically a U-bottom multiwell plate) that is subjected to appropriate surface treatment to reduce adhesion of cells involves operations that are simpler than those of the HD method. For example, Japanese Patent Application Laid-open No. 2004-254622 describes a culture vessel with a U-shaped bottom that is modified to be suitable for culturing ES cells, this method being an improvement over the aforementioned method. However, when culturing is performed using such a vessel with a U-shaped bottom, the cell mass is difficult to form and the culturing interval increases (that is, the growth rate of culture cells is low).